Various methods have been known for the determination of CPK activity. For example, Japanese Patent Publication No. 9988/71 corresponding to U.S. Pat. No. 3,540,984 discloses a method according to the scheme as shown below, wherein adenosine triphosphate (hereinafter ATP) is formed from CPK using creatine phosphoric acid (hereinafter "CP") and adenosine diphosphate (hereinafter ADP) as substrates. The ATP thus formed is converted into glucose 6-phosphoric acid (hereinafter "G6P") by utilizing glucose and hexokinase. The G6P thus formed is further converted into NADH or NADPH by using nicotinamide adenine dinucleotide (hereinafter NAD.sup.+) or nicotinamide adenine dinucleotide phosphate (heinafter) NADP.sup.+) and glucose 6-phosphate dehydrogenase (hereinafter "G6PDH"). An increase in NADH or NADPH is optically measured at a wavelength of 340 nm to thereby determine the CPK enzyme activity. ##STR1##
This method is widely known as the SSCC method. This method, however, needs the measurement of light in the ultraviolet region and thus suffers from disadvantages in that the equipment employed is expensive and the measurement is interferred by many compounds having an absorption in the ultraviolet region.
If a colorimetric method is employed wherein a formazane dye, having an absorption spectrum in the visible light region, is derived from the NADH formed by the enzymatic reaction, by utilizing an electron receiving dye precursor and an electron transfer agent, NADH can be quantitatively determined by spectral measurement in the visible light region. This method is described in, for example, Japanese Patent Application (OPI) No. 11395/74 corresponding to U.S. Pat. No. 3,663,374. The term "OPI" as used herein means a "published unexamined patent application".
The quantitative determination in an aqueous solution system needs a complicated procedure and a long amount of time because a series of chemical reactions must proceed in aqueous solution. In view of these problems, a dry analytical element has been developed which does not need a complicated liquid preparation process and which permits a rapid and simplified analysis. In particular, a mono-unit type multi-layer analytical element which is of high analytical accuracy is disclosed in, for example, Japanese Patent Publication No. 21677/78 corresponding to U.S. Pat. No. 3,992,158 and Japanese Patent Application (OPI) No. 164356/80 corresponding to U.S. Pat. No. 4,292,272. An attempt to incorporate the aforementioned reaction system for use in a CPK activity determination into the mono-unit type multi-layer analytical element has been made, as described in Japanese Patent Application (OPI) Nos. 88097/84 and 254199/86 corresponding to U.S. Pat. No. 4,713,327. However, in the storage stability of the above CPK activity determination elements in the summer season or at high temperatures in the tropics is insufficiently low. For example, when allowed to stand at 35.degree. C. for two days or more, the elements produce an error in the measurement of the enzymatic activity and thus are unsuitable for practical use.